Methods and compositions for detecting rhinoviruses
The invention provides methods and compositions for rapid, sensitive, and highly specific nucleic acid-based (e.g., DNA based) detection of human rhinovirus (HRV) in a sample. In general, the methods involve detecting a target nucleic acid having a target sequence of a conserved 5′ untranslated region of the HRV genome. The invention also features compositions, including primers, probes, and kits, for use in the methods of the invention.
1. A kit comprising oligonucleotides, wherein said oligonucleotides consist essentially of a first degenerate nucleic acid primer set, a second degenerate nucleic acid primer set, and a degenerate nucleic acid probe set, wherein said first primer set, said second primer set, and said probe set are each at least 8 nucleotides in length and are capable of binding under high stringency conditions to nucleic acid having the target sequence set forth in SEQ ID NO: 12, or a full complement thereof, wherein use of the primers and probe are capable of distinguishing human rhinovirus (HRV) from non-HRV picornaviruses.
2. The kit of claim 1 , wherein said first primer set comprises two or more sequences of the degenerate primer sequence set forth in SEQ ID NO: 8 or a full complement thereof.
3. The kit of claim 2 , wherein said two or more sequences of the first primer set comprise at least one primer sequence set forth in SEQ ID NO: 16, or a full complement thereof.
4. The kit of claim 1 , wherein said kit comprises at least one nucleic acid probe comprising the sequence set forth in SEQ ID NO: 10, or a full complement thereof.
5. The kit of claim 4 , wherein said probe comprises the sequence set forth in SEQ ID NO: 18.
6. The kit of claim 1 , wherein said kit comprises a nucleic acid probe that selectively binds human rhinovirus nucleic acids but not enterovirus nucleic acids.
7. The kit of claim 1 , wherein said second primer set comprises two or more sequences of the degenerate primer sequence set forth in SEQ ID NO: 9 or a full complement thereof.
8. The kit of claim 7 , wherein said two or more sequences of the second primer set comprise at least one primer sequence set forth in SEQ ID NO: 17, or a full complement thereof.
9. The kit of claim 1 , wherein said first primer set comprises two or more sequences of the degenerate primer sequence set forth in SEQ ID NO: 8 or a complement thereof, and wherein said second primer set comprises two or more sequences of the degenerate primer sequence set forth in SEQ ID NO: 9 or a full complement thereof.
10. The kit of claim 1 , wherein said probe set comprises a detectable label.
11. The kit of claim 10 , wherein said detectable label comprises a fluorescent label.
12. The kit of claim 11 , wherein said probe set further comprises a quenching moiety.
13. A kit comprising oligonucleotides, wherein said oligonucleotides consist essentially of a first degenerate nucleic acid primer set, a second degenerate nucleic acid primer set, and a degenerate nucleic acid probe set, wherein said first primer set, said second primer set, and said probe set are each at least 8 nucleotides in length and are capable of binding under high stringency conditions to nucleic acid having the target sequence set forth in SEQ ID NO: 1, or a full complement thereof, wherein use of the primers and probe are capable of distinguishing human rhinovirus (HRV) from non-HRV picornaviruses.
14. The kit of claim 13 , wherein said first primer set comprises two or more sequences of the degenerate primer sequence set forth in SEQ ID NO: 8 or a full complement thereof.
15. The kit of claim 13 , wherein said two or more sequences of the first primer set comprise at least one primer sequence set forth in SEQ ID NO: 16, or a full complement thereof.
16. The kit of claim 13 , wherein said second primer set comprises two or more sequences of the degenerate primer sequence set forth in SEQ ID NO: 9 or a full complement thereof.
17. The kit of claim 13 , wherein said two or more sequences of the second primer set comprise at least one primer sequence set forth in SEQ ID NO: 17, or a full complement thereof.
18. The kit of claim 13 , wherein said kit comprises at least one nucleic acid probe comprising the sequence set forth in SEQ ID NO: 10, or a full complement thereof.
19. The kit of claim 18 , wherein said probe comprises the sequence set forth in SEQ ID NO: 18.
20. The kit of claim 13 , wherein said kit further comprises a nucleic acid probe that selectively binds human rhinovirus nucleic acids but not enterovirus nucleic acids.
21. The kit of claim 13 , wherein said first primer set comprises two or more sequences of the degenerate primer sequence set forth in SEQ ID NO: 8 or a full complement thereof, and wherein said second primer comprises two or more sequences of the degenerate primer sequence set forth in SEQ ID NO: 9 or a complement thereof.
22. The kit of claim 13 , wherein said probe set comprises a detectable label.
23. The kit of claim 22 , wherein said detectable label comprises a fluorescent label.
24. The kit of claim 23 , wherein said probe set further comprises a quenching moiety.
25. The kit of claim 1 , wherein said non-HRV picornaviruses are enteroviruses.
26. The kit of claim 25 , wherein the enteroviruses are echoviruses, enterovirus 71, coxsackie viruses, or polioviruses.
27. The kit of claim 1 , wherein the primer sets and probe sets are suitable for use in a real-time PCR assay.
28. The kit of claim 13 , wherein the related said non-HRV picornaviruses viruses are enteroviruses.
29. The kit of claim 28 , wherein the enteroviruses are echoviruses, enterovirus 71, coxsackie viruses, or polioviruses.
30. The kit of claim 13 , wherein the primer sets and probe sets are suitable for use in a real-time PCR assay.